搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

In the adult bone marrow (BM),endothelial cells (ECs) are an integral component of the hematopoietic stem cell (HSC)-supportive niche,which modulates HSC activity by producing secreted and membrane-bound paracrine signals. Within the BM,distinct vascular arteriole,transitional,and sinusoidal EC subtypes display unique paracrine expression profiles and create anatomically-discrete microenvironments. However,the relative contributions of vascular endothelial subtypes in supporting hematopoiesis is unclear. Moreover,constitutive expression and off-target activity of currently available endothelial-specific and endothelial-subtype-specific murine cre lines potentially confound data analysis and interpretation. To address this,we describe two tamoxifen-inducible cre -expressing lines,Vegfr3-creER T2 and Cx40-creER T2,that efficiently label sinusoidal/transitional and arteriole endothelium respectively in adult marrow,without off-target activity in hematopoietic or perivascular cells. Utilizing an established mouse model in which cre -dependent recombination constitutively-activates MAPK signaling within adult endothelium,we identify arteriole ECs as the driver of MAPK-mediated hematopoietic dysfunction. These results define complementary tamoxifen-inducible creER T2 -expressing mouse lines that label functionally-discrete and non-overlapping sinusoidal/transitional and arteriole EC populations in the adult BM,providing a robust toolset to investigate the differential contributions of vascular subtypes in maintaining hematopoietic homeostasis. The online version contains supplementary material available at 10.1007/s12015-024-10703-9. View Publication

In this report,we describe a modification of the assay for long-term culture-initiating cells (LTC-IC) that allows a subset of murine LTC-IC (designated as LTC-ICML) to express both their myeloid (M) and lymphoid (L) differentiative potentials in vitro. The modified assay involves culturing test cells at limiting dilutions on irradiated mouse marrow feeder layers for an initial 4 weeks under conditions that support myelopoiesis and then for an additional week under conditions permissive for B-lymphopoiesis. All of the clonogenic pre-B progenitors (colony-forming unit [CFU] pre-B) detected in such postswitch LTC appear to be the progeny of uncommitted cells present in the original cell suspension because exposure of lymphoid-restricted progenitors to myeloid LTC conditions for textgreater or = 7 days was found to irreversibly terminate CFU-pre-B production and,in cultures initiated with limiting numbers of input cells (no progenitors of any type detected in textgreater 70% of cultures 1 week after the switch),the presence of CFU-pre-B was tightly associated with the presence of myeloid clonogenic cells,regardless of the purity of the input population. Limiting dilution analysis of the proportion of negative cultures measured for different numbers of input cells showed the frequency of LTC-ICML in normal adult mouse marrow to be 1 per 5 x 10(5) cells with an enrichment of approximately 500-fold in the Sca-1+ Lin-WGA+ fraction,as was also found for competitive in vivo repopulating units (CRU) and conventionally defined LTC-IC. LTC-ICML also exhibited the same resistance to treatment in vivo with 5-fluorouracil (5-FU) as CRU and LTC-IC,thereby distinguishing these three populations from the great majority of both in vitro clonogenic cells and day 12 CFU-S. The ability to quantitate cells with dual lymphoid and myeloid differentiation potentials in vitro,without the need for their prior purification,should facilitate studies of totipotent hematopoietic stem cell regulation. View Publication

Male germline stem cells (mGSCs) are stem cells present in male testis responsible for spermatogenesis during their whole life. Studies have shown that mGSCs can be derived in vitro and resemble embryonic stem cells (ESCs) properties both in the mouse and humans. However,little is know about these cells in domestic animals. Here we report the first successful establishment of goat GSCs derived from 2-5-month fetal testis,and developmental potential assay of these cells both in vitro and in vivo. These cells express pluripotent markers such as Oct4,Sox2,C-myc,and Tert when cultured as human ESCs conditions. Embryoid bodies (EBs) formed by goat mGSCs were induced with 2 × 10(-6) M retinoic acid (RA). Immunofluorescence analysis showed that some cells inside of the EBs were positive for meiosis marker-SCP3,STRA8,and germ cell marker-VASA,and haploid marker-FE-J1,PRM1,indicating their germ cell lineage differentiation. Some cells become elongated sperm-like cells after induction. Approximately 34.88% (30/86) embryos showed cleavage and four embryos were cultured on murine fibroblast feeder and formed small embryonic stem like colonies. However,most stalled at four-cell stage after intracytoplasmic sperm injection (ICSI) of these cells. Transplantation of DAPI labeled mGSCs into the seminiferous tubules of busulfan-treated mice,and showed that mGSCs can colonize,self-renew,and differentiate into germ cells. Thus,we have established a goat GSC cell line and these cells could be differentiated into sperm-like cells in vivo and sperms in vitro,providing a promising platform for generation of transgenic goat for production of specific humanized proteins. View Publication

Self-renewal is a complex biological process necessary for maintaining the pluripotency of embryonic stem cells (ESCs). Recent studies have used global proteomic techniques to identify proteins that associate with the master regulators Oct4,Nanog and Sox2 in ESCs or in ESCs during the early stages of differentiation. Through an unbiased proteomic screen,Banf1 was identified as a Sox2-associated protein. Banf1 has been shown to be essential for worm and fly development but,until now,its role in mammalian development and ESCs has not been explored. In this study,we examined the effect of knocking down Banf1 on ESCs. We demonstrate that the knockdown of Banf1 promotes the differentiation of mouse ESCs and decreases the survival of both mouse and human ESCs. For mouse ESCs,we demonstrate that knocking down Banf1 promotes their differentiation into cells that exhibit markers primarily associated with mesoderm and trophectoderm. Interestingly,knockdown of Banf1 disrupts the survival of human ESCs without significantly reducing the expression levels of the master regulators Sox2,Oct4 and Nanog or inducing the expression of markers of differentiation. Furthermore,we determined that the knockdown of Banf1 alters the cell cycle distribution of both human and mouse ESCs by causing an uncharacteristic increase in the proportion of cells in the G2-M phase of the cell cycle. View Publication

Regulated cell death is a key component of the innate immune response,which provides the first line of defense against infection and homeostatic perturbations. However,cell death can also drive pathogenesis. The most well-defined cell death pathways can be categorized as nonlytic (apoptosis) and lytic (pyroptosis,necroptosis,and PANoptosis). While specific triggers are known to induce each of these cell death pathways,it is unclear whether all cell types express the cell death proteins required to activate these pathways. Here,we assessed the protein expression and compared the responses of immune and non-immune cells of human and mouse origin to canonical pyroptotic (LPS plus ATP),apoptotic (staurosporine),necroptotic (TNF-α plus z-VAD),and PANoptotic (influenza A virus infection) stimuli. When compared to fibroblasts,both mouse and human innate immune cells,macrophages,expressed higher levels of cell death proteins and activated cell death effectors more robustly,including caspase-1,gasdermins,caspase-8,and RIPKs,in response to specific stimuli. Our findings highlight the importance of considering the cell type when examining the mechanisms regulating inflammation and cell death. Improved understanding of the cell types that contain the machinery to execute different forms of cell death and their link to innate immune responses is critical to identify new strategies to target these pathways in specific cellular populations for the treatment of infectious diseases,inflammatory disorders,and cancer. View Publication

Epithelial to mesenchymal transitions (EMTs) are key events during embryonic development and cancer progression. It has been proposed that Src plays a major role in some EMT models,as shown by the overexpression of viral Src (v-Src) in epithelial cells. It is clear that Src family kinases can regulate the integrity of both adherens junctions and focal adhesions; however,their significance in EMT,especially in the physiological context,remains to be elucidated. Here we showed that Src is activated in transforming growth factor-beta1 (TGF-beta1)-mediated EMT in mammary epithelial cells and that the Src family kinase inhibitor,PP1,prevents EMT. However,neither a more specific Src family kinase inhibitor,SU6656,nor a dominant-negative Src inhibited TGF-beta1-mediated EMT,leading us to speculate that Src activation is not an essential component of TGF-beta1-mediated EMT. Unexpectedly,PP1 prevented Smad2/3 activation by TGF-beta1,whereas SU6656 did not. Most interestingly,an in vitro kinase assay showed that PP1 strongly inhibited the TGF-beta receptor type I,and to a lesser extent,the TGF-beta receptor type II. Taken together,our data indicated that PP1 interferes with TGF-beta1-mediated EMT not by inhibiting Src family kinases but by inhibiting the Smad pathway via a direct inhibition of TGF-beta receptor kinase activity. View Publication

An essential step for therapeutic and research applications of stem cells is the ability to differentiate them into specific cell types. Endodermal cell derivatives,including lung,liver,and pancreas,are of interest for regenerative medicine,but efforts to produce these cells have been met with only modest success. In a screen of 4000 compounds,two cell-permeable small molecules were indentified that direct differentiation of ESCs into the endodermal lineage. These compounds induce nearly 80% of ESCs to form definitive endoderm,a higher efficiency than that achieved by Activin A or Nodal,commonly used protein inducers of endoderm. The chemically induced endoderm expresses multiple endodermal markers,can participate in normal development when injected into developing embryos,and can form pancreatic progenitors. The application of small molecules to differentiate mouse and human ESCs into endoderm represents a step toward achieving a reproducible and efficient production of desired ESC derivatives. View Publication

Early erythroid progenitors are transit-amplifying cells with high proliferative capacity committed to undergoing red cell differentiation. CD71/CD24low progenitors are less mature and have greater proliferative capacity than CD71/CD24high. We present protocols for isolation of CD71/CD24low progenitors from mouse fetal liver using both fluorescence-activated cell sorting (FACS) and immunomagnetic enrichment. CD71/CD24low progenitors isolated with both approaches show similar transcriptomes at single-cell resolution and exhibit characteristic proliferative responses to glucocorticoids. For complete details on the use and execution of this protocol,please refer to Li et al. (2019). View Publication

Large scale purification of endothelial cells is of great interest as it could improve tissue transplantation,reperfusion of ischemic tissues and treatment of pathologies in which an endothelial cell dysfunction exists. In this study,we describe a novel genetic approach that selects for endothelial cells from differentiating embryonic stem (ES) cells. Our strategy is based on the establishment of ES-cell clones that carry an integrated puromycin resistance gene under the control of a vascular endothelium-specific promoter,tie-1. Using EGFP as a reporter gene,we first confirmed the endothelial specificity of the tie-1 promoter in the embryoid body model and in cells differentiated in 2D cultures. Subsequently,tie-1-EGFP ES cells were used as recipients for the tie-1-driven puror transgene. The resulting stable clones were expanded and differentiated for seven days in the presence of VEGF before puromycin selection. As expected,puromycin-resistant cells were positive for EGFP and also expressed several endothelial markers,including CD31,CD34,VEGFR-1,VEGFR-2,Tie-1,VE-cadherin and ICAM-2. Release from the puromycin selection resulted in the appearance of alpha-smooth muscle actin-positive cells. Such cells became more numerous when the population was cultured on laminin-1 or in the presence of TGF-beta1,two known inducers of smooth muscle cell differentiation. The hypothesis that endothelial cells or their progenitors may differentiate towards a smooth muscle cell phenotype was further supported by the presence of cells expressing both CD31 and alpha-smooth muscle actin markers. Finally,we show that purified endothelial cells can incorporate into the neovasculature of transplanted tumors in nude mice. Taken together,these results suggest that application of endothelial lineage selection to differentiating ES cells may become a useful approach for future pro-angiogenic and endothelial cell replacement therapies. View Publication

Dysregulation of the receptor tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) plays a pathogenic role in a number of human hematopoietic malignancies and solid tumors. These include t(4;14) multiple myeloma associated with ectopic expression of FGFR3 and t(4;12)(p16;p13) acute myeloid leukemia associated with expression of a constitutively activated fusion tyrosine kinase,TEL-FGFR3. We recently reported that FGFR3 directly tyrosine phosphorylates RSK2 at Y529,which consequently regulates RSK2 activation. Here we identified Y707 as an additional tyrosine in RSK2 that is phosphorylated by FGFR3. Phosphorylation at Y707 contributes to RSK2 activation,through a putative disruption of the autoinhibitory alphaL-helix on the C terminus of RSK2,unlike Y529 phosphorylation,which facilitates ERK binding. Moreover,we found that FGFR3 interacts with RSK2 through residue W332 in the linker region of RSK2 and that this association is required for FGFR3-dependent phosphorylation of RSK2 at Y529 and Y707,as well as the subsequent RSK2 activation. Furthermore,in a murine bone marrow transplant assay,genetic deficiency in RSK2 resulted in a significantly delayed and attenuated myeloproliferative syndrome induced by TEL-FGFR3 as compared with wild-type cells,suggesting a critical role of RSK2 in FGFR3-induced hematopoietic transformation. Our current and previous findings represent a paradigm for tyrosine phosphorylation-dependent regulation of serine-threonine kinases. View Publication

In all,78 peripheral hematopoietic progenitor cell collections from 52 patients were evaluated using our previously published validated post-thaw assays at the time of collection and following transplantation by assessment of viable CD34(+) cells,and granulocyte-macrophage colony-forming units (CFU-GM) cryopreserved in quality control vials. The median (range) post-thaw recovery of viable CD34(+) cells and CFU-GM was 66.4% (36.1-93.6%) and 63.0% (28.6-85.7%),respectively,which did not show significant correlation with the engraftment of either neutrophils (P=0.136 and 0.417,respectively) or platelets (P=0.88 and 0.126,respectively). However,the reinfused viable CD34(+) cells/kg of patient weight pre- or post-cryopreservation showed significant correlation to engraftment of neutrophils (P=0.0001 and 0.001,respectively) and platelets (P=0.023 and 0.010,respectively),whereas CFU-GM pre- or post-cryopreservation was significantly correlated to neutrophils (P=0.011 and 0.007,respectively) but not to platelets (P=0.112 and 0.100,respectively). The results show that post-cryopreservation assessment of viable CD34(+) cells or CFU-GM is as reliable a predictor of rapid engraftment as that of pre-cryopreservation measures. Therefore,the post-cryopreservation number of viable CD34(+) cells or CFU-GM should be used to eliminate the risks of unforeseen cell loss that could occur during cryopreservation or long-term storage. View Publication